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From 2014.igem.org

Proof of Principle

Microscopy

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Agglutination Assay

We were able to successfully achieve the results we wanted with the Agglutination Assay. When antibodies are in the presence of their matching antigen pairs, they will bind to each other. Antibodies have two antigen binding sites which allows a network of antibody-antigen expressing bacteria to form. This network manifests on a large scale as a mat of bacteria on the bottom of the containing vessel, as opposed to a solid dot formed by unagglutinated antibodies. We based our assay off of this paper.

For our proof of principle agglutination, we used surface-bound YFP(Yellow Fluourescent Protein) and a corresponding GFP(Green Fluourescent Protein)/YFP antibody (the part of the protein that the antibody binds to is not affected by the part that causes the color change.) We used both of the surface expression proteins we had at our disposal: BclA (the one we made) and INP (a pre-existing biobrick). As controls, we used internally expressed YFP as well as a protein that was extrnally expressed but did not match with the antibody (we used the CAEV protein we made).

This photo clearly shows that the wells containing the bacteria with the matching antigen on the surface formed agglutinated mats while the control wells did not agglutinate and formed dots at the bottom of the wells.